BIOLCHEM 415 1st Edition Lecture 5 Outline of Last Lecture I. Hierarchial Protein Structure: primary, secondary, tertiary, quaternaryII. Secondary structures: α-helices and β-sheetsIII. Protein folding is driven by the hydrophobic effectIV. Disulfide bonds stabilize protein structureV. Protein misfolding can contribute to diseasesOutline of Current Lecture VI. Proteins can be purified according to their different physical propertiesVII. Gel electrophoresis can be used to analyze proteinsVIII. Antibodies can be used in protein identification and purificationCurrent LectureProtein Purification- protein concentration ~ 300 mg/mL in cellDifferential Contrifugation- first step- disrupt membrane -> organelles and cytoplasmic proteins- organelles all have different massesPurification Techniques- salting out: relative solubility (Chromatography more popular technique)- gel filtration chromatography: separation by size- ion exchange chromatography: separation by charge- affinity chromatography: purification based on different surface structures and ligand binding- high pressure liquid chromatography (HPLC): high resolution purificationSeparation based on solubilityThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- ammonium sulfate commonly used- different proteins precipitate at different ammonium sulfate concentrations - centrifuged to precipitate the proteins- dialysis- separates the salt and the protein- proteins suspended in a buffer in dialysis bag- pores in the bag allow ammonium sulfate to diffuse outGel Filtration Chromatography- separation based on size- beads in the filtration column have different sized pores- catch different sized proteinsIon-Exchange Chromatography- separation based on charge- positively charged proteins bond to the negatively charged beadsAffinity Chromatography- separation based on surface structures or ligand binding- small molecules attached to the beads- some proteins will bind to these molecules- others will be washed offHigh Pressure Liquid Chromatography- high pressure allows for more finely divided beads than typical columns- these have superior resolving powerAnalyzing Proteins by Gel Electrophoresis- Polyacrylamide Gel Electrophoresis (PAGE)- separation by size and charge- separation of proteins in their native state- SDS-PAGE- electrophoresis under denaturing conditions- Sodium dodecyl sulfate (SDS)- a denaturing agent- stain required for visualization- Isoelectric Focusing- gel has a gradient of high to low pH- voltage applied to gel- proteins move until they reach a pH in which they have a net charge of 0- 2D Gel Electrophoresis- proteins separated in two directions by two techniques1) Isoelectric focusing2) SDS-PAGE- proteins analyzed using mass spectroscopy Using Antibodies to Purify and Characterize Proteins - antibodies can be generated against protein epitopes- antibodies can bind to proteins with high affinities and specificity- polyclonal: recognize different epitopes- monoclonal: antibody isolated from antibody producing cell- Western blot can be used to detect a specific protein on a gelProtein Sequencing- amino acid sequence -> protein function- sequence initially was found using complete acid hydrolysis- later Edman degradation used- now either mass spectrometry or DNA sequencing is usedMass Spectrometry- measures mass/charge ratioX-ray Crystallography- image of 3D protein structure- ~3Å resolution needed to visualize amino acids and side chains- hydrogen needs 1+Å in order to be seenNMR spectroscopy- Nuclear magnetic resonance- used to determine structures of proteins, DNA, and RNA- generates multiple structures for a protein- performed in a solution - dynamic information- multiple structures create experimental uncertainty - more than 1 possible
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